The relative contributions of store-operated and voltage-gated Ca2+ channels to the control of Ca2+ oscillations in airway smooth muscle

Abstract

Key points: Agonist-dependent oscillations in the concentration of free cytosolic calcium are a vital mechanism for the control of airway smooth muscle contraction and thus are a critical factor in airway hyper-responsiveness. Using a mathematical model, closely tied to experimental work, we show that the oscillations in membrane potential accompanying the calcium oscillations have no significant effect on the properties of the calcium oscillations. In addition, the model shows that calcium entry through store-operated calcium channels is critical for calcium oscillations, but calcium entry through voltage-gated channels has much less effect. The model predicts that voltage-gated channels are less important than store-operated channels in the control of airway smooth muscle tone.

Abstract: Airway smooth muscle contraction is typically the key mechanism underlying airway hyper-responsiveness, and the strength of muscle contraction is determined by the frequency of oscillations of intracellular calcium (Ca²⁺ ) concentration. In airway smooth muscle cells, these Ca²⁺ oscillations are caused by cyclic Ca²⁺ release from the sarcoplasmic reticulum, although Ca²⁺ influx via plasma membrane channels is also necessary to sustain the oscillations over longer times. To assess the relative contributions of store-operated and voltage-gated Ca²⁺ channels to this Ca²⁺ influx, we generated a comprehensive mathematical model, based on experimental Ca²⁺ measurements in mouse precision-cut lung slices, to simulate Ca²⁺ oscillations and changes in membrane potential. Agonist-induced Ca²⁺ oscillations are accompanied by oscillations in membrane potential, although the membrane potential oscillations are too small to generate large Ca²⁺ currents through voltage-gated Ca²⁺ channels, and thus have little effect on the Ca²⁺ oscillations. Ca²⁺ entry through voltage-gated channels only becomes important when the cell is depolarized (e.g. by a high external K⁺ concentration). As a result, agonist-induced Ca²⁺ oscillations are critically dependent on Ca²⁺ entry through store-operated channels but do not depend strongly on Ca²⁺ entry though voltage-gated channels.

Keywords: airways; calcium dynamics; calcium influx; calcium signalling.

Figure 1. Schematic diagram of the model

Agonist binding to G‐protein‐coupled receptors stimulates the production of InsP₃, which diffuses into the cytosol. Calcium is released from the SR through IPR and RyR. Reuptake is via ATPase pumps (SERCA). Calcium effux is via the plasma membrane ATPase (PM) and influx is through ROCC, SOCC and L‐type VGCC Ca²⁺ channels. The model includes Ca²⁺‐activated K⁺ and Cl⁻ channels (KCa) and (ClCa), the delayed rectifier K⁺ currents (Kdr), basal Na⁺ (bNa) and K⁺ currents (bK) and the Na⁺‐K⁺ exchanger (NaK). Calcium fluxes and currents are denoted by J, whereas currents of Na⁺ and K⁺ are denoted by I. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2. Agonist‐induced Ca²⁺ oscillations and reduction of Ca²⁺ influx

A, model simulation of Ca²⁺ oscillations stimulated by [InsP₃] = 0.05 μM in the absence of extracellular Ca²⁺. During the oscillations, the cell loses Ca²⁺ and the oscillations cease after 30 s. B, the cell is subjected to the same stimulus and then the SOCC are blocked. Blocking SOCC stops the oscillations after ∼100 s. C, experimental results. Removal of extracellular Ca²⁺ results in disappearance of the oscillations after ∼40 s. D, blockage of SOCC by GSK‐7975A results in disappearance of the oscillations after ∼80 s. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3. Agonist‐induced Ca²⁺ oscillations and blocking of VGCC

A, an agonist binding to the G‐coupled receptor leads to the production of InsP₃. The simulations are performed with [InsP₃] = 0.05 μM. The period is similiar to the experimental results (B and C). Blocking VGCC by nifedipine leads to slower oscillations with the period halved. B, oscillations triggered by 400 nM MCh followed by blocking voltage‐gated Ca²⁺ entry by 100 μM nifedipine. C, the first 30 s show fast oscillations with periods of ∼1 s. D, nifedipine slows down the oscillations to a period of ∼2 s. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4. Depolarization‐induced Ca²⁺ oscillations and blocking of VGCC

A, simulations of oscillations in the Ca²⁺ concentration by depolarization with 50 mM KCl. Blocking VGCC, in the model, leads to an abrupt stop of Ca²⁺ oscillations and the plateau of Ca²⁺ concentration decreases back to baseline. B, intracellular Ca²⁺ oscillations in mouse airway smooth muscle cells induced by depolarization with 50 mM KCl. Depolarization causes elevated Ca²⁺ entry through VGCC and leads to overfilling of internal stores. The result is periodic calcium‐induced Ca²⁺ release with periods of ∼30 s. C, subsequently blocking VGCC by 10 μM nifedipine causes the oscillations to stop abruptly. The elevated cytosolic Ca²⁺ is extruded and the Ca²⁺ concentration slowly returns to values close to the resting state before depolarization. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 5. Depolarization‐induced Ca²⁺ oscillations and blocking of SOCC

A, the model predicts that blockage of SOCC causes a slight decrease in the frequency of KCl‐induced oscillations, with no other significant effects. B, experimental test of the model prediction. Blockage of SOCC with GSK7975A has little effect on KCl‐induced oscillations, although there is no obvious decrease in oscillation frequency. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 6. Model simulations of currents through plasma membrane Ca²⁺ channels for different stimuli

A, shows the currents of SOCC, ROCC, VGCC and the Ca²⁺ concentration in the SR ([Ca²⁺]_SR) for depolarization‐induced oscillations (50 mM KCl). B, shows the same currents for agonist‐induced oscillations and [Ca²⁺]_SR ([InsP₃] = 0.05 μM). Note the log scale for the currents. C, shows the effect of blocking VGCC by nifedipine on the current through SOCC during agonist‐induced oscillations. Note the different time scale, and the small oscillations in J _SOCC, which are caused almost entirely by the small oscillations in membrane potential (and thus in the driving force for Ca²⁺ current) that accompany the Ca²⁺ oscillations. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 7. Effect of voltage oscillations on agonist‐induced and depolarization‐induced Ca²⁺ oscillations

A, simulations of agonist‐induced ([InsP₃] = 0.05 μM) oscillations in [Ca²⁺] and V. B and C, Comparison of models with oscillating voltage (blue curves) vs. fixed voltage near the maximum (V ₀ = −43 mV; red curves). B, resting state (dashed lines) and the maximum of the Ca²⁺ concentration during oscillations (solid lines) for different values of InsP₃ concentrations. Thick lines indicate stable solutions and thin lines indicate unstable solutions. C, frequency as a function of InsP₃. D, simulations of KCl‐induced oscillations in [Ca²⁺] and V. Amplitude (E) and frequency (F) of KCl‐induced Ca²⁺ oscillations are shown in the presence of voltage oscillations (blue curves: dashed lines are steady‐state, solid lines denote the maximum [Ca²⁺] during an oscillation) and for a fixed voltage of the same average value (red curves). [Colour figure can be viewed at wileyonlinelibrary.com]